Depletable peroxidase-like activity of Fe3O4 nanozymes accompanied with separate migration of electrons and iron ions

As pioneering Fe3O4 nanozymes, their explicit peroxidase (POD)-like catalytic mechanism remains elusive. Although many studies have proposed surface Fe2+-induced Fenton-like reactions accounting for their POD-like activity, few have focused on the internal atomic changes and their contribution to the catalytic reaction. Here we report that Fe2+ within Fe3O4 can transfer electrons to the surface via the Fe2+-O-Fe3+ chain, regenerating the surface Fe2+ and enabling a sustained POD-like catalytic reaction. This process usually occurs with the outward migration of excess oxidized Fe3+ from the lattice, which is a rate-limiting step. After prolonged catalysis, Fe3O4 nanozymes suffer the phase transformation to γ-Fe2O3 with depletable POD-like activity. This self-depleting characteristic of nanozymes with internal atoms involved in electron transfer and ion migration is well validated on lithium iron phosphate nanoparticles. We reveal a neglected issue concerning the necessity of considering both surface and internal atoms when designing, modulating, and applying nanozymes.

Since the first discovery of Fe 3 O 4 nanoparticles (NPs) with intrinsic peroxidase (POD)-like activity in 2007, nanomaterial-based artificial enzymes (nanozymes) and their extensive applications have rapidly attracted attention over the past decade [1][2][3][4][5][6][7][8][9][10] . Recently, research efforts on nanozymes have gradually shifted from application-oriented to mechanism-oriented [11][12][13][14][15] . For example, single-atom nanozymes centered on different metal species have been synthesized with welldefined structures and coordination environments, which facilitate the identification of catalytic centers and unravel the catalytic mechanisms at the atomic level [16][17][18][19] . Besides, the high substrate selectivity of nanozymes has been achieved by the bionic principle of natural substrate channeling and stepwise screening or by molecular blotting techniques 20,21 . Given the intricate structure-activity relationships and restricted characterization techniques, however, it is still challenging to understand the explicit mechanism of most nanozymes 3,4 .
Despite being a pioneering nanozyme, research on the catalytic mechanism of the POD-like activity of Fe 3 O 4 NPs is still limited 10,14,15,[22][23][24][25] . To date, it is generally accepted that high-reactive hydroxyl radicals (·OH) generated by Fenton-like reactions (Eqs. (1) and (2)) involving the surface Fe 2+ under acid conditions contribute to the POD-like activity of Fe 3 O 4 NPs 24,25 . Although the questioning that the POD-like activity of magnetite is mediated by adventitious metal traces, it has been experimentally verified (including this paper) to exclude the interference of metal traces or the leaching effect of Fe ions in reaction solution 10,[25][26][27][28] . Similar to natural horseradish peroxidase, Fe 3 O 4 nanozymes follow the ping-pong mechanism and Michaelis-Menten kinetics 10 . In addition, their catalytic performances are influenced by particles size, morphology, lattice structure, doping, surface modification, substrates used, and the catalytic environment exposed, all of which could affect the surface-active sites by altering the surface chemistry 10,22,23,[29][30][31][32] . Other individual studies have investigated the absorption, activation, and desorption processes of substrates (e.g., H 2 O 2 and 3, 3′, 5, 5′-tetramethylbenzidine (TMB)) on the surface of Fe 3 O 4 at the atomic level based on density functional theory and developed some descriptors to predict their POD-like activity 14 The above mechanistic studies share a theoretical premise: only the surface-active sites play a decisive role in the enzymatic-like property of nanozymes since catalysis occurs mainly on the particle surface or interface. This view is now widely recognized and works for most types of nanozymes 1,2,4,11,32,33 . For example, in a recent controversial question regarding how to define nanozyme concentration, Liu et al. argued that considering the whole particle or all atomic units within a particle as an enzyme unit would overestimate and underestimate the catalytic activity of nanozymes, respectively, because it is the surface atoms that are truly the catalytic active sites 33 . However, in the Fenton-like reactions triggered by Fe 3 O 4 nanozymes, we noticed that the reaction rate constant of Eq. (1) is much higher than that of Eq. (2), which implies that the surface-active Fe 2+ is hardly recovered after being oxidized. This irreversible oxidation of surface Fe 2+ prompts us to ponder if only the surface atoms of the nanozymes, particularly for metal oxide nanozymes, act in enzymatic-like catalysis, would these active sites be exhausted after long-term catalysis, rendering the nanozymes inactive? Nevertheless, no relevant studies can conclusively answer this crucial question.
Here, we propose a neglected issue regarding the POD-like mechanism of nanozymes by characterizing the chemical composition and catalytic activity of the recycled Fe 3 O 4 NPs participating in cyclic POD-like catalysis. Both surface and interior Fe 2+ are found to impart POD-like properties to Fe 3 O 4 nanozymes. Generally, Fe 2+ inside the particle could transfer its electron to the surface layer, regenerating the surface Fe 2+ and sustaining the catalytic reaction. This process is usually coupled with the outward migration of excess oxidized Fe 3+ , which is probably a rate-limiting step. As the catalysis continues, Fe 3 O 4 is slowly oxidized into γ-Fe 2 O 3 accompanying the depleted enzymelike activity, similar to the conventional low-temperature oxidation of magnetite, only with different electron receptors. This self-depleting characteristic of nanozymes with internal atoms involved in electron transfer and ion migration is further demonstrated by a typical model material, lithium iron phosphate (LiFePO 4 ), which contains redoxactive metal sites and mobile lithium ions (Li + ) encapsulated in a rigid phosphate network. This paper reveals that internal atoms may also contribute to nanozyme-catalyzed reactions even though these reactions occur on the surface of NPs, which is thought-provoking when designing, regulating, and applying nanozymes.

Synthesis and characterization of IONPs
Near-spherical magnetite nanoparticles (Fe 3 O 4 NPs) with an average diameter of 10.16 ± 0.12 nm (Supplementary Fig. 1a) were synthesized using the chemical coprecipitation method 24 . Maghemite (γ-Fe 2 O 3 ) and hematite (α-Fe 2 O 3 ) NPs were derived by calcining the Fe 3 O 4 NPs powder at 200 and 650°C for 2 h, respectively (Fig. 1a) 34 . X-ray diffractometer (XRD) and Raman spectra ( Supplementary Fig. 1b, c) show the successful synthesis of these three iron oxide NPs (IONPs). These IONPs were uniformly dispersed in an aqueous solution at pH of 3 by ultrasonication ( Supplementary Fig. 1d). Their POD-like activities were assessed using different colorimetric substrates, including TMB, 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and o-Phenylenediamine (OPD), in the presence of H 2 O 2 . The results show that their catalytic activity followed the order of Fe 3 O 4 NPs >> γ-Fe 2 O 3 NPs > α-Fe 2 O 3 NPs ( Supplementary Fig. 2). To better quantify their POD-like activity, we calculated their specific activity (a nano ) according to the specified method 35,36 , which were 1.79, 0.45, and 0.03 U·mg −1 , respectively (Fig. 1b). As previously reported 10,24 , the higher catalytic ability of Fe 3 O 4 NPs is attributed to the ·OH arising from the surface Fe 2+ -initiated Fenton-like reaction ( Supplementary Figs. 3 and 4). The negligible a nano of α-Fe 2 O 3 NPs compared with γ-Fe 2 O 3 NPs is ascribed to the structural effect of the crystal phases 37 . Briefly, γ-Fe 2 O 3 possess cation vacancies at its octahedral positions and most of these vacancies are located on the particle surface, which can favor the adsorption of the substrate H 2 O 2 , resulting in a relatively higher POD-like activity ( Supplementary Fig. 5). However, these vacancies do not exist on the surface of α-Fe 2 O 3 due to the change of crystal structure caused by the higher calcination temperature 37 .

Cyclic POD-like catalysis of Fe 3 O 4 NPs
To investigate whether the surface Fe 2+ of Fe 3 O 4 NPs is depleted after participating in prolonged catalysis, we continuously increased the amount of substrate TMB under sufficient H 2 O 2 with as-synthesized three IONPs as continuous catalysts, and monitored the absorbance changes of TMB oxidation products at 650 nm within 12 h. From Supplementary Fig. 6d-g, even though the TMB was increased from 0.087 to 0.52 mM, the Fe 3 O 4 NPs were still able to continuously and rapidly engage in the catalytic reaction for a long duration (≥12 h) without showing signs of depletion. We speculated two reasons: (1) the amount of substrate is still too low to completely consume the surfaceactive Fe 2+ and (2) the Fe 2+ within Fe 3 O 4 NPs provides the impetus for continuous catalysis.
Cyclic POD-like catalytic assays (Fig. 1c) were carried out as validation, which could provide sufficient substrates for Fe 3 O 4 NPs to keep exerting their POD-like capacity. We evaluated the a nano of the recycled Fe 3 O 4 NPs within 5 days. The results show that the catalytic ability of Fe 3 O 4 NPs decreased to a level comparable to that of γ-Fe 2 O 3 NPs after five days of cyclic catalysis, while the changes of γ-Fe 2 O 3 NPs were negligible ( Fig. 1d and Supplementary Fig. 7). The impact of leached Fe ions in acidic medium on the catalytic activity of IONPs has been excluded (Supplementary Fig. 8 and Supplementary Table 1). It pushed us to wonder how the surface-active Fe 2+ of Fe 3 O 4 NPs alone could sustain the TMB oxidation for up to 100 h. Conceivably, if only the surface-active sites are responsible for the enzyme-like performance, nanozymes will deactivate when the surface-active sites are exhausted ( Supplementary Fig. 9).
To reveal the potential reasons for the sustained catalytic capacity of Fe 3 O 4 NPs, we characterized the physicochemical properties of the recycled Fe 3 O 4 NPs using different methodologies. The electron energy-loss spectra are a useful tool for revealing the chemical and oxidation state information of iron oxide at high spatial resolution 38 . In general, the peaks of the transition metal L-edge shift toward higher energy loss with an increasing oxidation state. For iron oxide species, the area ratios of Fe L 3 /L 2 also increase with increasing Fe valence 39 . As shown in Fig. 1e, both of the Fe 3 O 4 NPs before and after 5 days of cyclic POD-like reactions showed two peaks related to the Fe L 3 and L 2 , with an energy gap of about 13 eV between the two white lines. However, approximately 1.4 eV chemical shift toward high energy loss could be observed from Fe 3 O 4 NPs to recycled Fe 3 O 4 NPs. In addition, the Fe L 3 / L 2 area ratios also increased from 4.7 to 6.1, which indicates the increase in the Fe oxidation state of Fe 3 O 4 NPs after 5 days of POD-like catalysis 40 . A similar finding was obtained from the XPS analysis of the recycled Fe 3 O 4 NPs on days 0, 1, 3, and 5 of the cyclic catalysis. The X-ray penetration depth of the analyzed sample ranges from 2 to 10 nm for the XPS technique. Since the average diameter of as-synthesized Fe 3 O 4 NPs is around 10 nm, the Fe valence state obtained from the Fe2p fitting analysis can be approximated as the oxidation state of individual Fe 3 O 4 NPs. As shown in Fig. 1f, the Fe 2+ in Fe 3 O 4 NPs decreased from the original 30.9% to 0% with the extension of cyclic catalytic days, suggesting that the interior Fe 2+ may also be oxidized to Fe 3+ in the successive POD-like reactions.
The phase transformation of Fe 3 O 4 NPs caused by internal Fe 2+ oxidation was demonstrated by the near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. Figure 1g shows the Fe L-edge   [41][42][43] . Besides, in the Raman spectra of the recycled Fe 3 O 4 NPs, the feature of the A 1g mode band gradually shifted from 660 to 700 cm −1 (Fig. 1h), corresponding to a transition from magnetite to maghemite 44,45 . In addition, transmission electron microscopy (TEM) images ( Fig. 1i) and XRD pattern (Supplementary Fig. 10) show that the influence of this transformation on the particle morphology, size, and lattice structure is negligible.
Based on the above characterization results, we conclude that not only the surface Fe 2+ but also the interior Fe 2+ of the Fe 3 O 4 nanozymes were gradually oxidized by prolonging the reaction time. Simultaneously, the catalytic activity of the recovered NPs gradually decreases with the increase of their oxidation state. Therefore, we suggest that the involvement of Fe 2+ inside the particles is responsible for the prolonged catalytic capacity of Fe 3 O 4 nanozymes. Specifically, as shown in Supplementary Fig. 11, when the surface Fe 2+ is oxidized to Fe 3+ by the Fenton-like reaction, the adjacent Fe 2+ inside the particle will continuously transfer its electron outward via the Fe 2+ -O-Fe 3+ chain in the lattice to maintain the catalytic activity of the surface Fe atoms. However, this replenishment of electrons is not infinite. When all the interior Fe 2+ are oxidized to Fe 3+ , the Fe 3 O 4 phase is transformed to γ-Fe 2 O 3 without electrons being transferred to the surface, resulting in the reduction of catalytic activity or even inactivation.

Aeration oxidation kinetics of Fe 3 O 4 NPs
We assume that the oxidation of Fe 3 O 4 nanozymes induced by PODlike catalysis is comparable to the traditional low-temperature (<200°C) air oxidation of magnetite since the crystal structure of both remains unchanged during the oxidation process 46  Lattice defects have been reported to facilitate the outward migration of excess iron ions, thereby accelerating the oxidation process of magnetite 48 . As verification, we compared the aeration oxidation kinetics of Fe 3 O 4 NPs synthesized by two methods with different degrees of lattice defects. One was prepared by the chemical coprecipitation method as described above (Fig. 1a), which is considered to possess more lattice defects (named cc-Fe 3 O 4 NPs). The other was prepared by the thermal decomposition method (Supplementary Fig. 12) with a relatively complete lattice structure (named TD-Fe 3 O 4 NPs) 50 . Both Fe 3 O 4 NPs have a similar average particle size (~10 nm) with N(CH 3 ) 4 + as a surface stabilizer. Their aqueous solutions were stirred under the same aeration rate (with air) for 12 h at 120°C. For a better comparison, the oxidation system of cc-Fe 3 O 4 NPs (total 170 mL, 3.6 mg Fe/mL) was much larger than that of TD-Fe 3 O 4 NPs (total 30 mL, 0.45 mg Fe/mL). This implies that individual TD-Fe 3 O 4 could gain more oxygen than cc-Fe 3 O 4 to keep it oxidized. From Fig. 2a, b, both Fe 3 O 4 NPs exhibited electronic transitions in the visible and NIR region due to intervalence charge transfer between Fe 2+ and Fe 3+ 51 , which decreased gradually with oxidation time. At the end of aeration oxidation, little absorption beyond 700 nm was observed, indicating a phase transformation from Fe 3 O 4 NPs to γ-Fe 2 O 3 NPs 51 . Besides, the color of both suspensions gradually changed from darkbrown to reddish-brown. Notably, despite the less oxygen exposure for individual cc-Fe 3 O 4 NP, its NIR absorption decreased faster than that of TD-Fe 3 O 4 NP, especially during the initial oxidation phase (within 1 h). These results confirm that more lattice defects favor the oxidation reaction of Fe 3 O 4 NPs due to the faster electron and ion transfer.
Analogous to aerated oxidation, the rapid electron and ion migration are also considered to facilitate the POD-like catalysis of Fe 3 O 4 NPs, with the only difference being that the electron receptor changed from O 2 in aerated oxidation reaction to H 2 O 2 in POD-like reaction. To prove this, the POD-like activity of cc-Fe 3 O 4 NPs and TD-Fe 3 O 4 NPs as well as their variation with aerated oxidation time were investigated. As seen in Supplementary Fig. 13, the POD-like activity of cc-Fe 3 O 4 NPs was higher (2.8 folds) than that of TD-Fe 3 O 4 NPs, despite TD-Fe 3 O 4 NPs having a smaller hydrodynamic diameter and negative surface potential contributing to a strong affinity with TMB. Aeration oxidation kinetic studies show that the POD-like activity of both Fe 3 O 4 NPs decreased with oxidation time (Fig. 2c), along with slight fluctuations in hydrodynamic size and surface potential ( Supplementary  Fig. 14). However, the decline rate of cc-Fe 3 O 4 NPs was faster than TD-Fe 3 O 4 NPs, particularly in the initial oxidation stage. This phenomenon is consistent with the changes in NIR spectra shown in Fig. 2a, b. These results further confirm that the more lattice defects of Fe 3 O 4 NPs, the easier the migration of excess Fe ions, and thus the higher the POD-like activity. It also means that Fe 3 O 4 NPs with more defect sites are easier to be depleted when involved in a POD-like reaction due to their excellent catalytic capability.

Catalytic mechanism of the POD-like for Fe 3 O 4 NPs
Taken together, the catalytic mechanism of the POD-like activity for NPs is thought to be analogous to the conventional low-temperature air oxidation of magnetite, in which iron ion migration is probably a ratelimiting step.
As is known, magnetite has an inverse spinel structure, with Fe 3+ occupying tetrahedral (A) sites and equal amounts of Fe 2+ 54 , each vacancy is electrically equivalent to an extra −5/2 charge at one B-site, which has to be neutralized by the excess positive charge at the adjacent B-sites. Thus, each vacancy traps 5 Fe 3+ and no longer involves in the conduction process 53 . In general, this disturbed electron-hopping process caused by the reduction of Fe 2+ -Fe 3+ pairs and the formation of cation vacancies is thought to impair the electron transfer to the surface when Fe 3 O 4 nanozymes participate in the sustained POD-like reaction, leading to their depletable catalytic activity.

LiFePO 4 NPs as an ideal verification model
To test the above mechanism, we found an ideal model material, LiFePO 4 , which is commonly applied as cathode material for lithiumion batteries (LIBs) [55][56][57][58] . LiFePO 4 undergoes redox reactions along with the lithium insertion/extraction during the charge-discharge process (Eqs. (5) and (6)) without changing its ordered-olivine structure (Fig. 4a) 55 . We speculate that the charging process of LiFePO 4 resembles the oxidation process of Fe 3 O 4 , both of which involve the oxidation of Fe 2+ and the migration of internal ions, which motivated us to focus on whether LiFePO 4 NPs also have the POD-like catalytic ability.
Rod-like LiFePO 4 NPs with an average length of 321.9 nm and width of 172.2 nm (Fig. 4b) were successfully synthesized using the solvothermal method 59 and characterized via various methodologies (Supplementary Fig. 15 and Supplementary Tables 2 and 3). As expected, the POD-like activity of LiFePO 4 NPs was demonstrated with different chromogenic substrates including TMB, ABTS, and OPD ( Fig. 4c and Supplementary Fig. 16). Also, they follow pH, temperature as well as NPs concentration dependence, and the Michaelis-Menten kinetics ( Supplementary Figs. 17 and 18). The optimal pH is about 4.0. The ·OH was shown to be generated in a time-and concentrationdependent manner (Fig. 4d and Supplementary Fig. 19), which is similar to Fe 3 O 4 NPs. We then compared the POD-like activity of LiFePO 4 NPs and cc-Fe 3 O 4 NPs using two oppositely charged substrates (TMB and ABTS) at pH 3.6. The results consistently show that LiFePO 4 NPs had higher catalytic ability than cc-Fe 3 O 4 NPs (Supplementary Fig. 20), and the a nano of LiFePO 4 NPs was approximately four times than that of cc-Fe 3 O 4 NPs, despite their larger particle size (Fig. 4e). These results imply that LiFePO 4 NPs may share a similar PODlike catalytic mechanism with Fe 3 O 4 NPs, differing in that the rapid Li + migration in the lattice of LiFePO 4 NPs confers them a superior PODlike catalytic activity (Fig. 4f).

Phase transformation of LiFePO 4 NPs
The recycled LiFePO 4 NPs from three cycles of POD-like catalysis were proven to suffer a phase transformation to FePO 4 via multiple characterization techniques. Specifically, the XPS Fe2p peaks of the Article https://doi.org/10.1038/s41467-022-33098-y recycled LiFePO 4 NPs were shifted toward the higher binding energy (Fig. 5a), indicating the oxidation of Fe 2+ within the NPs. In the XRD pattern (Fig. 5b), the residual LiFePO 4 phase (marked with о in the yellow pattern) in the recycled NPs was negligible, proving that almost all LiFePO 4 were delithiated and oxidized into FePO 4 (marked with +) after cyclic POD-like catalysis. This result was further confirmed by ICP analysis that the Li element content in recycled NPs was almost 0 ( Table 1). This phase transformation, as expected, severely impaired the POD-like activity of the recycled LiFePO 4 NPs (Fig. 5c), in agreement with the self-depleting characteristic of the Fe 3 O 4 NPs described above.
As a LIBs cathode material, the reversible lithiation and delithiation characteristic of LiFePO 4 contribute to its superior electrochemical performance. We measured the cyclic voltammograms (CV) of the as-prepared LiFePO 4 NPs before and after participating in the cyclic POD-like reactions under different scan rates in the electrolyte containing Li + or Na + . As shown in Supplementary Fig. 21, the increase in redox peak currents (I p ) was proportional to the square root of scan rate (v 1/2 ), implying a diffusion-controlled process of Li + or Na + extraction and insertion 60 . Noticeably, the I p of the recycled LiFePO 4 NPs (i.e., FePO 4 ) was obviously reduced compared to LiFePO 4 NPs (Fig. 5d), especially in the electrolyte containing Na + (Supplementary Fig. 21e-h), indicating that the presence of mobile Li + contributes significantly to the redox peak current. As a comparison with LiFePO 4 NPs, the CV curves of Fe 3 O 4 NPs were also measured under the same conditions. As shown in Supplementary Fig. 22, the I p also exhibits a linear relation with the v 1/2 . However, unlike LiFePO 4 NPs, the I p of the recycled Fe 3 O 4 NPs (i.e., γ-Fe 2 O 3 ) only showed a marginal decrease compared to their counterparts before participating in the catalytic reaction, both of which were found to be similar to the I p of recycled LiFePO 4 NPs under the same scanning rate (Supplementary Fig. 22 and Fig. 5d). This is probably explained by the lack of freely diffusing ions in the lattice of iron oxide and FePO 4 , which weakens the electron transfer process in their redox reactions. By contrast, LiFePO 4 NPs exhibited the highest I p , ascribed to the availability of Li ions in their crystals.

Mobile Li-ions as the limiting factor
In the field of sodium (Na)-ion batteries, the charge transfer resistances and lattice volume change upon Na + migration are larger for NaFePO 4 electrodes, compared with their Li equivalents due to the larger ionic radius of Na (1.02 Å) than Li (0.76 Å) 61 . Inspired by this, We partially replaced Li with Na in the lattice of LiFePO 4 NPs to explore the potential effect of Na-doping on their POD-like activity. Concretely, three NaLiFePO 4 NPs with similar physicochemical properties but different Na-doping amounts were successfully synthesized (Supplementary Fig. 23 and Supplementary Table 4). We then compared their POD-like activities under the same reaction conditions and found that the more Na doping, the lower the POD-like activity (Fig. 5e), indicating that the large Na + radius hinders the free migration of Na + and Li + in the crystal, thereby impairing the electron transfer rate. We attempted to use K-doped LiFePO 4 NPs as further proof, however, the large ionic radius of K (1.38 Å) makes it difficult to embed into the electrode materials ( Supplementary Fig. 24 and Supplementary Table 4), which is a common issue in K-ion batteries 62 .
To further prove the decisive role of mobile Li + , we measured the POD-like activity of commercially available LiFePO 4 , Fe 3 (PO 4 ) 2 , and FePO 4 materials with similar hydrodynamic dimensions and surface negative potentials (Supplementary Fig. 25). The results show that their POD-like activity follows LiFePO 4 >> Fe 3 (PO 4 ) 2 > FePO 4 (Fig. 5f), directly confirming that the presence of Fe 2+ alone in Fe 3 (PO 4 ) 2 cannot ensure the superior catalytic performance, but the transportable Li + contributes to the outstanding POD-like activity of LiFePO 4 .
In summary, the catalytic mechanism of the POD-like activity of Fe 3 O 4 nanozymes is elucidated by characterizing the chemical composition and catalytic activity of the Fe 3 O 4 NPs recycled from the long-term POD-like catalysis. These studies indicate that not only the surface Fe 2+ , but also the internal Fe 2+ contribute to the POD-like activity of Fe 3 O 4 nanozymes. The Fe 2+ inside the particle can transfer electrons to the surface, regenerating the surface Fe 2+ that is constantly involved in the sustained catalytic reaction. This process is usually accompanied by the outward migration of excess oxidized Fe 3+ from the interior of the crystal, which is considered as a rate-limiting step. Analogous to the lowtemperature oxidation of magnetite, Fe 3 O 4 NPs participated in the POD-like reaction are eventually oxidized to γ-Fe 2 O 3 NPs with a reduced POD-like capacity. Furthermore, this mechanism is wellvalidated on LiFePO 4 NPs. This work reveals the depletable property of Fe 3 O 4 nanozymes differing from natural enzymes and highlights the potential contribution of internal metal atoms in nanozymes-catalyzed reactions. Meanwhile, these findings provide a theoretical basis for the mechanistic study and rational design of other related nanozymes. was purchased from Shanghai Maclin Biochemical Technology Co., Ltd. Lithium iron phosphate (LiFePO 4 ) was purchased from Shanghai Xushuo Biological Technology Co., Ltd. All chemicals were used as received without further purification. Deionized water was used throughout the experiments.

Characterization
The particle size and morphology were observed by scanning electron microscopy (SEM, Ultra Plus, Carl Zeiss, Germany). TEM, selective area electron diffraction (SAED), and high-resolution TEM (HRTEM) were taken using transmission electron microscopy (TEM, JEOL JEM-2100F, Japan). The phase and crystalline structure were explored by XRD (Brucker D8 Advance, Germany) and the DIFFRAC plus XRD Commander software (v2.6.1), using Cu Kα radiation. The element composition, chemical structures, and properties of NPs were analyzed by SEM energy dispersive spectroscopy (EDS), ICP-OES coupled with ICP Expert II software (Agilent ICPOES730), Fourier transform infrared spectroscopy coupled with OMNIC software v9.6 (FTIR, Nicolet IS 10, Thermo, USA), Raman spectrometer coupled with WiRE™ software v2.0  (Renishaw Invia, UK) and X-ray photoelectron spectroscopy coupled with Avantage software v5.967 (XPS, Thermo ESCALAB 250Xi, USA). The chemical speciation of Fe in IONPs was determined by NEXAFS at the beamline of 08U1A in the Shanghai Synchrotron Radiation Facility, Shanghai, China. The NEXAFS spectra of the references including FeSO 4 and Fe 2 O 3 were also measured. Hydrodynamic diameters and Zeta potential of particles were measured by the dynamic light scattering (DLS, Nano-ZS90, Malvern, England) and Malvern Zetasizer software (v7.12). The UV-Vis-NIR absorption spectra were measured using UV3600 (Shimadzu, Japan) or Microplate Reader (Infinite M200, Tecan, Switzerland). CV was measured using Electrochemical Workstation (CHI760e) and CHI-760E electrochemical software (v20.04). The production of ·OH was detected by the electron spin resonance spectrometer (ESR, Bruker EMXplus, Germany) and Bruker Xenon software (v1.2) at ambient temperature.

Synthesis of IONPs by chemical coprecipitation method
The naked magnetite NPs were synthesized by a modified chemical coprecipitation method, namely, cc-  34 . Finally, these three powders were dissolved in a pH of 3 aqueous solutions by ultrasound and stored at 4°C for subsequent experiments.

Synthesis of Fe 3 O 4 NPs by thermal decomposition method
Firstly, the OA-coated Fe 3 O 4 NPs were synthesized according to the thermal decomposition method (denoted as TD-Fe 3 O 4 @OA NPs) 50 . The chloroform solvent was removed from 4 mL of TD-Fe 3 O 4 @OA NPs (1.627 mg Fe/mL) by rotary evaporation. Then, the OA layer on the particle surface was replaced with 15 mL of 0.5% TMAOH under ultrasonic stirring 63 . TMAOH is a phase transfer small molecule agent which can form an electrostatic double layer on the particle surface and stabilize the particles in the aqueous phase. Next, the reaction system was transferred to 60 mL of the separatory funnel, and 45 mL of chloroform was added. The mixture was thoroughly mixed. After standing for 15 min, the underlying oil phase liquid was discarded. This extraction procedure was repeated three times to fully remove the free OA that was replaced from the particle surface by TMAOH. Finally, the obtained Fe 3 O 4 NPs aqueous solution (denoted as TD-Fe 3 O 4 NPs) was filtered through a 0.22-μm filter and stored at 4°C for subsequent experiments.

Synthesis of LiFePO 4 NPs and Na-or K-doped LiFePO 4 NPs
LiFePO 4 NPs were successfully synthesized by glycol-based solvothermal method 59 . In a typical route, 0.27 mL H 3 PO 4 (85% wt) was dropped into 9 mL of EG containing 0.365 g of LiOH·H 2 O. The mixture was stirred thoroughly until the neutralization reaction was complete, showing a milk-white suspension. Then 9 mL of EG dissolved 0.890 g FeSO 4 ·7H 2 O was added into the suspension under vigorous stirring. The mixtures slowly turned grayish-green. After stirring for 30 min, the reaction solution was transferred into a sealed hydrothermal reactor, and heated at 180°C for 10 h. At the end of the reaction, the final products were cooled down to room temperature and washed with deionized water three times.
For Na or K-doped LiFePO 4 NPs, the Li element content (8.69 mmol) of 1%, 5%, and 10% in the above method was replaced with Na element in Na 2 CO 3 or K element in KOH. The rest of the synthesis steps remained unchanged. Briefly, for Na-doped LiFePO 4 NPs, 0.365 g of LiOH·H 2 O was replaced with 4.6 mg of Na 2 CO 3

The POD-like activity of NPs
The POD-like activity of NPs was measured by different colorimetric substrates, including TMB, ABTS, and OPD, in the presence of H 2 O 2 under different reaction conditions. The absorbance of the colored oxidation products at the corresponding wavelength (TMB ox : 650 nm; ABTS ox : 415 nm; OPD ox : 492 nm) was monitored by a microplate reader and Tecan i-control software (v1.6.19.2). The effects of pH (2.5-11), temperature (10-65°C), and LiFePO 4 NPs concentration (0-6.25 μg Fe/mL) on POD-like activity were also studied.

Measurement of the specific activity (a nano ) of NPs
The POD-like specific activity of NPs synthesized in this work was determined according to the modified method specified in the national standard of China (GB/T 37966-2019) 35 . The following provided the general procedures: (a) monitor the temperature inside the quartz cuvette and incubate all reagents and samples to 25°C; (b) add 2.000 mL of 0.2 M acetate buffer solution (pH = 3.6) to a reaction container; (c) add 0.100 mL of different concentrations of NPs and 0.100 mL of 10 mg/mL of TMB in order, mix completely and incubate for 60 s at 25°C; (d) add 0.200 mL of 30% H 2 O 2 and mix completely; (e) immediately transfer the appropriate amount of reaction solution to the cuvette and monitor the changes of absorbance at 650 nm within the specified times using UV-Vis spectroscopy and UV Probe software (v2.42). The initial change rate of absorbance (min −1 ) is obtained from the slope of the early phase of time course; (f) measurement of reagent blank rate 1 and reagent blank rate 2: 30% H 2 O 2 and NPs are respectively replaced by deionized water. The measurement procedure is as described in Step a-e above; (g) after deducting the reagent blank rate 1 and 2, the POD-like catalytic activity units (b nano ) of NPs can be calculated according to Eq. (7): where b nano is the POD-like catalytic activity units of NPs (U); V is the total volume of reaction solution (μL); ΔA/Δt is the slope of the initial liner portion of absorbance changing over time after correcting with reagent blank rate 1 and 2 (min −1 ); ε is the molar absorption coefficient of TMB derivative (39,000 mol −1 ·L·cm −1 ); l is the optical path of the cuvette (cm). The specific activity of NPs (a nano ) can be calculated according to Eq. (8): where a nano is the specific activity of NPs (U·mg −1 ); m Fe is the total Fe element mass contained in added NPs (mg); 4b nano 4m Fe is the slope of the curve plotting the b nano against different masses of NPs (m Fe ).

ESR measurement
The production of ·OH was detected by adding 10 μg Fe/mL of cc-Fe 3 O 4 NPs or LiFePO 4 NPs and 30 mM of DMPO into 300 μL of acetate buffer (pH = 3.6) with or without 0.5% H 2 O 2 . The signal of the spin adduct (DMPO/·OH) was recorded at ambient temperature at the 1st, 5th, and 10th min after all reagents were mixed. The experimental parameters were as follows: 1 G modulation amplitude, 100 kHz modulation frequency, 6.325 mW microwave power, 9.829 GHz resonance frequency.
·OH detection using terephthalic acid as a fluorescent probe NPs were re-added into a new reaction system mentioned above to catalyze a new round of POD-like reactions. Totally, 5 rounds (or days) of catalysis were carried out, and each round lasted for 20 h at 25°C. The Fe 3 O 4 NPs recycled on days 0, 1, 3, and 5 were taken for further characterization. For LiFePO 4 NPs, 1 mL of LiFePO 4 NPs (8.4 mg Fe/mL) was added to a 20 mL of acetate buffer (pH 3.6) containing 1 mL TMB (10 mg/mL) and 2 mL of 30% H 2 O 2 . This catalytic reaction system lasted for 1 h at 25°C. After catalysis, LiFePO 4 NPs in the reaction solution were recycled by centrifugation, and then ultrasonic washed with deionized water several times. The recycled LiFePO 4 NPs were re-added into a new reaction system mentioned above to catalyze a new round of POD-like reactions. Totally, 3 rounds of catalysis were carried out, and each round lasted for 1 h at 25°C. The LiFePO 4 NPs recycled from the 0 and 3 rounds were taken for further characterization.

Aerated oxidation of Fe 3 O 4 NPs
A total of 170 mL of the as-synthesized cc-Fe 3 O 4 NPs suspensions (3.57 mg Fe/mL) was transferred into a 250 mL three-necked flask and heated from room temperature to 120°C. Air was pumped into the suspension at a constant rate (9 L/min) for 12 h under stirring (350 rpm). Condensation reflux was kept throughout the aeration oxidation. Samples oxidized at 0, 0.5, 1, 3, 5, 8 10, and 12 h were used for subsequent characterization.
As for TD-Fe 3 O 4 NPs, the only difference was that 30 mL of TD-Fe 3 O 4 NPs (0.45 mg Fe/mL) were transferred into a 100 mL threenecked flask. Other steps were consistent with the aeration oxidation of cc-Fe 3 O 4 NPs.

Electrochemical performance of NPs
The CV was used to evaluate the electrochemical performance of NPs in a three-electrode cell 35 . A total of 10 μL of NPs (100 μg Fe/mL) was mixed with 90 μL of 0.5% Nafion solution, and then dropped 30 μL of the above mixture into the polished glassy carbon electrode, which was dried and used as the working electrode. Mercury/mercurous sulfate (Hg/Hg 2 SO 4 ) was used as the reference electrode and platinum wire as the counter electrode. The electrolyte solution was 0.2 M lithium acetate or sodium acetate buffer (pH = 3.6). The CV was recorded at different voltage scan rates (ν).

Statistical and reproducibility
All experiments were repeated three times independently, and the data were presented as the mean values ± standard deviations (SD). Error bars shown in this paper represent the SD derived from three repeated measurements. UV probe Software v2.42, Tecan i-control Software v1.6.19.2, Malvern Zetasizer Software v7.12, FluorEssence Software v3.8, Bruker Xenon Software v1.2, CHI-760E electrochemical Software v20.04, WiRE™ 2.0 Software, Thermo Avantage Software v5.967, ICP Expert II Software, OMNIC Software v9.6, and DIFFRAC plus XRD Commander Software v2.6.1 were used for data collection. GraphPad Prism 8 and Origin 2018 were used for data processing and statistical analysis.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
All data supporting the findings of this study are available within the article and its Supplementary Information files. Data are available from the corresponding author upon request. Source Data are provided with this paper.